We report on the production of hydrocortisone, the major adrenal glucocorticoid of mammals and an important intermediate of steroidal drug synthesis, from a simple carbon source by recombinant Saccharomyces cerevisiae strains. An artificial and fully self-sufficient biosynthetic pathway involving 13 engineered genes was assembled and expressed in a single yeast strain. Endogenous sterol biosynthesis was rerouted to produce compatible sterols to serve as substrates for the heterologous part of the pathway. Biosynthesis involves eight mammalian proteins (mature forms of CYP11A1, adrenodoxin (ADX), and adrenodoxin reductase (ADR); mitochondrial forms of ADX and CYP11B1; 3beta-HSD, CYP17A1, and CYP21A1). Optimization involved modulating the two mitochondrial systems and disrupting of unwanted side reactions associated with ATF2, GCY1, and YPR1 gene products. Hydrocortisone was the major steroid produced. This work demonstrates the feasibility of transfering a complex biosynthetic pathway from higher eukaryotes into microorganisms.

Despite recent technical improvements, the construction of recombinant adenovirus vectors remains a time-consuming procedure which requires extensive manipulations of the viral genome in both Escherichia coli and eukaryotic cells. This report describes a novel system based on the cloning and manipulation of the full-length adenovirus genome as a stable plasmid in E. coli, by using the bacterial homologous recombination machinery. The efficiency and flexibility of the method are illustrated by the cloning of the wild-type adenovirus type 5 genome, the insertion of a constitutive promoter upstream from the E3 region, the replacement of the E1 region by an exogenous expression cassette, and the deletion of the E1 region. All recombinant viral DNAs were shown to be fully infectious in permissive cells, and the modified E3 region or the inserted foreign gene was correctly expressed in the infected cells.

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